Ultrastructural evidence for innervation of the endothelium and interstitial cell in the atrioventricular valves of the Japanese monkey.код для вставкиСкачать
THE ANATOMICAL RECORD 240:157-166 (1994) Ultrastructural Evidence for Innervation of the Endothelium and Interstitial Cells in the Atrioventricular Valves of the Japanese Monkey TOSHIKO TSUMORI AND TOKIO DOMOTO Department of Anatomy, Shimane Medical University, Izumo 693, Japan Background: A rich supply of nerves to the atrioventricular ABSTRACT valve has been demonstrated. The role of the valvular nerves is still controversial because the target sites of the nerves have not been confirmed. Methods: The innervation of the atrioventricular valves of the Japanese monkey (Macaca fuscata) was examined by acetylcholinesterase staining and electron microscopy. Immunoreactivity for neuropeptide Y (NPY) was also investigated by a post-embedding immunogold method. Results: The valvular nerve elements were clearly concentrated between the endothelium and interstitial cells on the atrial side of cusps. Naked axon terminals were observed to make direct contact (20-nm gaps) with interstitial cells and also to be in close proximity (-200-nm cleft) to the endothelium. NPY immunoreactivity was clearly detected on the large granular vesicles in some terminals that were in close proximity to interstitial cells andlor the endothelium. Conclusion: The present study suggests that the extensive innervation of the atrioventricular valve, which includes NPY-containing nerves, might affect valvular function via interstitial cells and/or the endothelium. 0 1994 Wiley-Liss, Inc. Key words: Atrioventricular valve. Nerve terminals, NeuroDeDtide Y. Interstitial cells, Electron microscopy, Immunogold staining, Japanese monkey A rich supply of neural elements to the atrioventric- Hibbs and Ellison (1973) reported that the valvular ular valves has been demonstrated not only in humans muscle fibers were not innervated directly, and that (Ferreira and Rossi, 1974; De Biasi et al., 1984; transmitter substances might diffuse to them from naKawano et al., 1989),but also in a variety of mammals ked nerve varicosities. De Biasi et al. (1984) proposed that includes guinea pigs, hedgehogs, rabbits, dogs, that smooth muscle bundles and blood vessels were the rats, cats, and pigs (Williams, 1964; Voloshchenko, most probable targets of the nerve fibers in the human 1965; Cooper e t al., 1966; Sonnenblick et al., 1967; Lipp and porcine valves. Thus, the possible terminal sites of and Rodin, 1968; Ehinger et al., 1969; Anderson, 1971; the valvular nerves have been the focus of some conSmith, 1971; Ellison and Hibbs, 1973; Hibbs and Elli- troversy. To clarify this issue, it is essential to obtain son, 1973; De Biasi et al., 1984; Kalman, 1988). More- ultrastructural evidence for the presence (or absence) over, recent immunohistochemical studies have re- of nerve terminals on the valve. vealed that valvular nerves contain substance P, We previously reported the ultrastructural localizavasoactive intestinal polypeptide, calcitonin gene-re- tion of NPY-like immunoreactivity (NPY-LI) within lated peptide, and neuropeptide Y (NPY) (Papka et al., axon varicosities contained in nerve bundles beneath 1981, 1984; Della et al., 1983; Kawano et al., 1989; the endothelium in the atrioventricular valves of the Zhang et al., 1993). However, the role of nerves in Japanese monkey (Zhang et al., 1993). However, no valve function remains obscure. It was suggested ini- direct evidence for the target sites of the axon varicostially that the valvular nerve fibers might serve a n ities that contained NPY-LI vesicles was provided by afferent function because the majority of these fibers the previous study. appeared to have no apparent terminal effector sites The aim of the present study was to characterize the within the valve (Williams, 1964; Voloshchenko, 1965; fine structure of nerve terminals and their target sites Anderson, 1971), while Cooper et al. (1966) proposed in the atrioventricular valves of the Japanese monkey that intravalvular neural elements might merely traverse the leaflets and be unrelated to valvular function. By contrast, Sonnenblick et al. (1967) reported that the cardiac muscle fibers in the canine mitral valve appeared to be under neural control. By contrast, Received August 30, 1993; accepted April 11, 1994 :c 1994 WILEY-LISS, INC 158 T. TSUMOKI AND T. DOMOTO and to detect NPY-LI on those nerve terminals by a n immunogold method. MATERIALS AND METHODS Four Japanese monkeys (Macaca fusucata; 4-6 kg) were used in this study. They were anesthetized by intramuscular injection of Ketalar (Sankyo Co. Ltd., Tokyo; 0.25 mlikg) and sacrificed by bleeding via the severed femoral artery. The hearts were rapidly removed and the mitral and tricuspid valves were dissected out and divided into several cusps. Acetylcholinesterase Staining For whole-mount preparations, the cusps were fixed in 4% neutral formalin for 30 min. After washing with phosphate buffered saline (PBS), tissues were incubated in a solution of acetylthiocholine iodide a s substrate by a modified version of the method of Karnovsky and Roots (1964), in the presence of 4 mM tetraisopropylpyrophosphoramide (iso-OMPA, Sigma Chemical Co., St. Louis, MO), for 3-4 h r a t room temperature. Then they were stretched on glass slides, airdried, covered with Entellan (Merck, Darmstadt, F.R. Germany), and examined. Some of the cusps were rapidly frozen in liquid nitrogen. Frozen sections (40 Fm) encompassing the entire layer of the cusps were cut on a cryostat and were collected in PBS. Sections were fixed in 4% neutral formalin for 5 min and then incubated a s described above. Electron Microscopy The cusps of valves were immersed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for about 10 min and then cut into small pieces. These pieces were fixed in the same fixative for a n additional 2 h r a t room temperature. After postfixation in 1%OsO, in 0.1 M cacodylate buffer for 1 h r at 4“C, tissues were dehydrated in a graded ethanol series, cleared in propylene oxide, and embedded in Epon 812. Thin sections, encompassing the entire layer of the valve cusps, were cut on a n ultramicrotome and mounted on copper grids. After staining with uranyl acetate and lead citrate, the sections were examined with a n electron microscope (JEOL JEM 1200 EX). lmmunogold Staining Thin sections were mounted on nickel grids and immunostained by a slightly modified version of the technique described by Bendayan and Zollinger (1983). The sections were etched with 3% hydrogen peroxide for 5 min and treated with a saturated aqueous solution of sodium metaperiodate for 15 min. The sections were preincubated with 1%ovalbumin in PBS for 30 min and then incubated with antiserum against NPY (Incstar Co., Stillwater, MINN; 1:200) for 60 min a t 30°C. After thorough rinsing with PBS, sections were incubated with gold-conjugated goat antibodies against rabbit IgG (Amersham International plc, Bucks., UK; 1 5 0 ) for 60 rnin a t 30°C. Finally, the sections were rinsed well with PBS and then with distilled water. Prior to electron microscopy, the sections were lightly stained with lead citrate and uranyl acetate. The specificity of the immunolabelling was examined by substi- Fig. 1. Acetylcholinesterase staining of a mitral valve cusp; wholemount sample. AChE-positive fibers form a fine mesh-like network. Bar: 40 km. tuting PBS, normal rabbit serum, or preabsorbed antiserum for the NPY-specific antiserum. No specific labelling was observed in control sections. RESULTS Light Microscopy Acetylcholinesterase (AChE) staining revealed a dense nerve plexus in the atrioventricular valve of Japanese monkey. In the whole-mount specimens, AChEpositive fibers formed a fine mesh-like network in both the mitral and tricuspid valves (Fig. 1).In cross-sections, AChE-positive fibers seemed to be concentrated beneath the endothelium that faced the atrium. Electron Microscopy On the atrial side of valve cusps, the thin layer consisting of connective tissue cells was observed beneath the endothelium (Fig. 2). These cells were described by Hibbs and Ellison (1973) as “interstitial cells” in their study of the guinea-pig valve. The valvular “interstitial cells” observed in the Japanese monkey were characterized by abundant cytoplasmic filaments and numerous surface pits on the cell membrane (Fig. 3).They were located singly or in rows, extending long, slender AX E F I LGV M N NT S scv SGV Abbreviations Axon Endothelium Filament Interstitial cell Large granular vesicle Mitochondrion Nerve bundle Neurotubule Schwann cell Small clear vesicle Small granular vesicle INNERVATION OF ATRIOVENTRICULAR VALVES Figs. 2-1 0. Electron micrographs of atrioventricular valves. Figs. 2 and 3. Fig. 2. The atrial side of a mitral valve cusp. Interstitial cells ( I ) are located beneath the endothelium ( E ) and extend long, slender cytoplasmic processes parallel to the endothelium. A nerve bundle ( N ) containing many axon varicosities is seen between 159 the processes of interstitial cells (indicated by arrows). There are numerous collagenous fibrils in the stroma. Bar: 2 km. Fig. 3. An example of part of the process of an interstitial cell in the mitral valve cusp. Note the numerous cytoplasmic filaments (indicated by arrowheads) oriented along the axis of the process. Several surface pits are seen on the plasma membrane (arrows).Bar: 200 nm. 160 T. TSUMORI A N D T. DOMOTO Figs. 4 and 5 161 INNERVATION OF ATRIOVENTRICULAR VALVES cytoplasmic processes parallel to the endothelium (Figs. 2,4; see also Fig. 6). Muscle elements were scarce in the stroma of valve cusps, although the specimens taken from the region attached to the atrioventricular ring contained cardiac muscle fibers. Neural elements in the valve cusp were concentrated just beneath the endothelium or among interstitial cells. The nerve fibers can be described by reference to their location and their relationship to other components. (1)There were nerve bundles that contained several (occasionally more than ten) axon varicosities and these bundles were located adjacent to interstitial cells (Fig. 4). Some axon varicosities within each bundle were incompletely ensheathed by a Schwann cell and were closely associated with interstitial cells; the axon membrane and the plasma membrane of such cells were separated by a cleft that was -200 nm wide (Fig. 5). (2) Some terminals, not covered by a Schwann cell (“naked terminals”), were in direct contact (20-nm gaps) with the plasma membrane of interstitial cells (Figs. 6, 7; see also Fig. 10). (3) Some terminals were adjacent to the endothelium but were consistently separated by a cleft of -200 nm (Figs. 8, 9). Such axons were sometimes connected to interstitial cells (Fig. 9). (4) There were also some terminals that were not obviously associated with any other components. The composition in terms of synaptic vesicle of axon varicosities or terminals was generally similar. Most axon varicosities contained small clear vesicles (SCV), small granular vesicles (SGV), and large granular vesicles (LGV). SCV and SGV of 30-60 nm in diameter were in the majority. LGV (60-120 nm in diameter) were round or oval, with cores of varying density (Figs. 5-9). Some terminals that were filled with many smaller mitochondria, but with only a few ordinary vesicles, were observed beneath the endothelium and between interstitial cells. These terminals were consistently connected with cytoplasmic processes of interstitial cells (Fig. 10). Axo-axonal contact was occasionally seen within nerve bundles or at terminals. In such cases, axon membranes were close to each other (20-nm gaps) but no specialized membrane structure was apparent. lmmunoelectron Microscopy NPY-LI was clearly detected in LGV in the axon varicosities or terminals (Figs. 11, 12). Immunolabelled nerve terminals were often found close to interstitial cells. Some of these terminals were in direct contact (20-nm gaps) with the plasma membrane of interstitial cells (Fig. 11).Immunolabelled terminals Figs. 4 and 5. The atrial side of a mitral valve cusp. Nerve bundles (N)containing many axon varicosities are seen adjacent to the processes of interstitial cells (I). Figure 5 shows the area in Figure 4 enclosed by a rectangle a t higher magnification (Bar: 2 Fm). Some axon varicosities are incompletely ensheathed by a Schwann cell (S) and are closely associated with an interstitial cell. Axon varicosities contain large granular vesicles (LGV), small granular vesicles (SGV), small clear vesicles (SCV), neurotubules (NT),and mitochondria (M). Numerous surface pits (indicated by arrows) and abundant cytoplasmic filaments (F) are seen in the process of an interstitial cell. Bar: 200 nm. were also seen just beneath the endothelium (Fig. 12). In such cases, they were usually separated by clefts of -500 nm in width. DISCUSSION Unlike the atrioventricular valves in other mammals (Williams, 1964; Cooper et al., 1966; Sonnenblick et al., 1967; Anderson, 1971; Ellison and Hibbs, 1973; Hibbs and Ellison, 1973; De Biasi e t al., 1984), those in Japanese monkeys seemed to have no obvious arrangement of cardiac muscle fibers or smooth muscle fibers in the distal portion of the valve cusps. Only a thin layer consisting of interstitial cells was recognized just beneath the endothelium on the atrial side but not on the ventricular side. In the present study, we obtained ultrastructural evidence that nerve fibers terminate in interstitial cells and in the endothelium in the atrioventricular valves of the Japanese monkey. The densely distributed nerves in the monkey’s valves may possibly play a role in the control of valvular function via interstitial cells and the endothelium. Our results support, in part, the observations made by Filip et al. (1986), who reported that motor nerve endings are located in close apposition to interstitial cells in the atrioventricular valves of several laboratory animals and humans. With respect to the role of the valvular interstitial cells, Filip e t al. (1986) presented evidence that the interstitial cells of heart valves have features similar to those of smooth muscle cells, as follows: they contain numerous actin filaments and dense bodies as well a s intermediate filaments; they contain cyclic GMP-dependent protein kinase; they are innervated; and they are able to undergo slow, sustained contractions in response to epinephrine and angiotensin 11. These observations led Filip et al. to postulate that interstitial cells can provide “a controlled tonus, actively correlated with the cyclically changing forces acting on valves during diastole and systole.” Interstitial cells might be as candidates for the contractile elements, replacing the muscle components in the monkey’s valves. We recently reported the distribution of NPY-containing nerve fibers in the atrioventricular valves of monkeys (Zhang et al., 1993). In the present study, we obtained, for the first time, a direct evidence that the axon terminals that contain LGV with NPY-LI make contact with interstitial cells and are in close proximity to the endothelium. It is noteworthy that the valvular interstitial cells, which might be similar in character to smooth muscle cells, were found to be innervated by NPY-containing nerves. It has been proposed that NPY might influence cardiac function via its ability to induce vasoconstriction and the pre-junctional release of noradrenaline (Wharton and Gulbenkian, 1987). Filip et al. (1986) reported that most of the nerve endings in close apposition to interstitial cells appeared to be of the adrenergic type. Further investigations are needed to ascertain whether noradrenaline and NPY coexist in the valvular nerves and to examine the pharmacological effects of NPY on valvular interstitial cells. Unlike the terminal sites of the NPY-containing nerves in the interstitial cells, axon varicosities and the endothelium were usually separated by relatively wide clefts (-500 pm). However, i t is possible that NPY released from the naked axon varicosities might 162 T. TSUMORI AND T. DOMOTO Figs. 6 and 7. Two examples of axon terminals in direct contact with the processes of interstitial cells (1) in the tricuspid valve. Junctional clefts between the axon membranes and the cytoplasmic membranes of interstitial cells are about 20 nm wide (indicated by arrowheads). Arrows indicate surface pits on the interstitial cell (I).Fig. 6. A small axon terminal is seen connecting with a process of an interstitial cell (1). Bar: 2 pm. The inset shows the area enclosed by a rectangle at higher magnification. LGV: Large granular vesicle; M: mitochondrion. Bar: 100 nm. Fig. 7. A large terminal contains numerous synaptic vesicles which include large granular vesicles (LGV), small granular vesicles (SGV),and small clear vesicles (SCV).Bar: 200 nm INNERVATION OF ATRIOVENTRICULAR VALVES Figs. 8 and 9. Axon terminals adjacent to the endothelium tE) on the atrial side of the tricuspid valve. Fig. 8. shows large, naked varicose terminals that contain numerous large granular vesicles (LGV), small granular vesicles (SGV), and small clear vesicles (SCV).Bar: 2 163 pm. The inset shows the area enclosed by a rectangle at higher magnification. Bar: 200 nm. Fig. 9. An axon terminal containing a large number of SCV and a few LGV makes close contact with the processes of interstitial cells (I).The arrow indicates a surface pit. Bar: 200 nm. 164 T. TSUMORI AND T. DOMOTO Fig. 10. An example of an axon terminal (in this case, from the tricuspid valve) with numerous mitochondria and flattened vesicles, but few normal vesicles. This terminal is completely surrounded by processes (arrows) of the interstitial cells (I). Junctional clefts be- tween the axon membrane and the plasma membrane of interstitial cells are about 20 nm wide (indicated by arrowheads). A normal, small axon terminal having a few small granular vesicles (SGV) is seen in close proximity to an interstitial cell. Bar: 200 nm. have an effect on the endothelium. The role of NPYcontaining nerves in the valvular endothelium, in particular on the atrial side, remains to be clarified. We cannot exclude the possibility that the nerve elements in the valves include sensory elements. We occasionally found axon varicosities that were filled with numerous mitochondria and a few ordinary vesicles. This type of axon varicosity is similar to those described by Hibbs and Ellison (1973) and De Biasi e t al. (1984) a s sensory endings. According to Furness and Costa (19801, axons with many small mitochondria may be sensory axons. Burnstock (1986) also proposed that axons packed with mitochondria and a few vesicles and surrounded by cell processes might be sensory corpuscles. With respect to the role of sensory neural elements, it has been proposed that the nerve network associated with the valves might be involved in the Figs. 11 and 12. Electron micrographs showing the ultrastructural localization of immunoreactivity for neuropeptide Y in axon terminals beneath the endothelium. The atrial side of a tricuspid valve. Fig. 11. An axon terminal (AX) in a direct contact with an interstitial cell (I). The areas enclosed by rectangles are shown a t higher magnification in (a) and (b). Bar: 1 km. a: The junctional cleft between the plasma membrane of an interstitial cell (I) and the axon membrane is indicated by arrowheads. An immunolabelled, large granular vesicle is seen (arrow)in the axon terminal. Bar: 100 nm. b: Arrows indicate immunolabelled, large granular vesicles. E: endothelium; M: mitochondrion. Bar: 100 nm. Fig. 12. Two axon varicosities (AX)just beneath the endothelium (E). An axon (AX with an arrow) is free from t.he Schwann cell (S) envelope on t.he side that faces the endothelium. The areas enclosed by rectangles are shown a t higher magnification in ( a ) and (b). Bar: 1 pm. a: An arrow indicates an immunolabelled, large granular vesicle. Small granular vesicles (SGV) are not labelled. M: mitochondrion; Bar: 100 nm. b Large granule vesicles (arrows) are immunolabelled. Small clear vesicles (SCV) are not labelled. Bar: 100 nm. INNERVATION OF ATRIOVENTRICULAR VALVES Figs. 11 and 12. 165 166 T. TSUMORI AND T. DOMOTO “measurement” of blood-flow parameters as the blood moves across the cusps (Williams, 1964) and with the “recording” of stretching of the cusps during the different phases of cardiac activity (Voloschenko, 1965). In conclusion, the dense supply of nerves in the atrioventricular valve may be closely related to valvular function via interstitial cells and the endothelium. NPY is a possible candidate for a neurotransmitter or a neuromodulator in valvular innervation. ACKNOWLEDGMENTS The authors are grateful to Prof. E.E. Daniel of McMaster University for a critical review of the manuscript, to Mrs. Yuki Kobayashi for technical assistance in the electron microscopy, and to Miss Chiho Mochida for typing the manuscript. This study was supported by Grant-in-Aid 05770010 for General Scientific Research from the Ministry of Education, Science, and Culture, Japan. LITERATURE CITED Anderson, R.H. 1971 Fluorescing valvular nerves in the guinea-pig heart. Experientia, 27: 1063-1064. Bendayan, M., and M. Zollinger 1983 Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J. Histochem. Cytochem., 31:101-109. 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